NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells
NaKtide, a Na/K-ATPase-derived Peptide Src Inhibitor, Antagonizes Ouabain-activated Signal Transduction in Cultured Cells
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DOI：
10.1074/jbc.m109.013821
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发表时间：
2009-07-31
影响因子：
4.8
通讯作者：
Xie, Zijian
中科院分区：
文献类型：
--
作者：
Li, Zhichuan;Cai, Ting;Xie, Zijian
我们之前已经证明Na/ k - atp酶结合并抑制Src。在这里，我们报道了Na/ k - atp酶介导的Src调控的分子机制，以及一种新型肽Src抑制剂的产生，该抑制剂靶向Na/ k - atp酶/Src受体复合物并拮抗瓦阿因诱导的蛋白激酶级联反应。首先，Na/ k - atp酶通过α - 1亚基核苷酸结合结构域的N端抑制Src激酶。其次，详细的定位导致鉴定出一个20个氨基酸的肽（NaKtide），它以ATP浓度无关的方式抑制Src （IC50 = 70 nM）。此外，NaKtide并不直接影响ERK和蛋白激酶C家族的激酶。其抑制Lyn的效价较低（IC50 = 2.5 μ M）。第三，包括HIV-Tat-NaKtide （pNaKtide）在内的高度带正电的前导肽偶联物很容易进入培养细胞。最后，pNaKtide的以下功能研究表明，该偶联物可以特异性靶向Na/ k - atp酶相互作用的Src池，并在培养细胞中作为一种有效的瓦巴因拮抗剂：1)与PP2不同，pNaKtide存在于膜中。与此一致的是，它对基础Src活性的影响远小于PP2。2) pNaKtide以剂量依赖性的方式有效地破坏Na/ k - atp酶/Src受体复合物的形成。因此，它阻断了瓦苦因诱导的Src、ERK活化和心肌细胞肥厚生长。3)与PP2不同，pNaKtide不影响igf诱导的心肌细胞ERK活化。综上所述，我们认为pNaKtide可以作为一种新的瓦巴因拮抗剂，用于探索Na/ k - atp酶和强心剂类固醇新发现的信号功能的生理和病理意义。
We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/K-ATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the alpha 1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC50 = 70 nM) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC50 = 2.5 mu M). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.